Neonatal necrotizing enterocolitis (NEC) is the single most devastating gastrointestinal cause of mortality and morbidity in premature infants. Although the pathophysiology of NEC is uncertain, the leading hypothesis is that intestinal bacteria penetrate the immature intestinal epithelial barrier defenses, leading to tissue invasion and subsequent necrosis. A key gastrointestinal cell of the innate immune system is the Paneth cell, which is located in the base of the intestinal crypts. Paneth cells protect the intestine through secretion of pro-inflammatory mediators and antimicrobial substances. Compared to age-matched controls, infants with NEC have significantly decreased numbers of Paneth cells. Despite the key role Paneth cells play in immune function, it is unclear what role these cells play in the pathogenesis of NEC. Traditional animal models have not yet developed Paneth cells, and are thus unable to address this gap in knowledge. The overall objective of this pre-clinical application is to determine the role of Paneth cells in the development of tissue injury in the immature intestine. To achieve this objective, we will utilize our newly described mouse model of NEC that uses Paneth cell necrosis to induce NEC-like injury. Our hypothesis is that Paneth cell loss renders the immature small intestine susceptible to development of NEC. In this application, we propose to test this hypothesis in the following two Aims: 1: Establish the mechanism of Paneth cell involvement in the pathogenesis of NEC. Infants with NEC lack Paneth cells, and Paneth cell ablation in the presence of Klebsiella produces NEC- like pathology in mice. However, the role of Paneth cells in intestinal injury remains unclear. In this Aim we will examine methods of Paneth cell loss, and the role of Paneth cell secretions to establish how Paneth cell disruption renders the immature ileum susceptible to injury. 2: Establish the role of TLR4 signaling in Paneth cell ablation-induced NEC. Bacteria are required for development of NEC, and TLR4 activation has been proposed as an initiator for disease development4. In the ileum where NEC occurs, TLR4 is expressed in the intestinal crypts. However, these studies were done in intestines that had not yet developed Paneth cells. Thus, it is unclear what role TLR4 plays in Paneth cell ablation-induced NEC. In this Aim we will examine the TLR4 signaling pathway in connection to Paneth cell disruption and subsequent injury. These proposed studies are innovative as they investigate a heretofore-unappreciated role for Paneth cells in the development of NEC. The results of this research will provide a clearer understanding of the mechanism of Paneth cell ablation-induced NEC in the immature intestine. This is the first important step needed for developing novel ways to predict disease development and new pharmacologic strategies to treat NEC.